Medical professionals and cytotechnologists often prepare biological specimens on a specimen carrier, such as a slide, and review specimens to analyze whether a patient has or may have a particular medical condition or disease. For example, it is known to examine a cytological specimen in order to detect malignant or pre-malignant cells as part of a Papanicolaou (Pap) smear test and other cancer detection tests. To facilitate this review process, automated systems focus the technician's attention on the most pertinent cells or groups of cells, while discarding less relevant cells from further review. One known automated imaging system that has been effectively used in the past is the ThinPrep Imaging System, available from Cytyc Corporation, 250 Campus Drive, Marlborough, Mass. 01752.
FIG. 1 generally illustrates one known biological screening system 10 that is configured for presenting a biological specimen 12 located on a microscope slide 14 (as shown in FIG. 2) to a technician, such as a cytotechnologist, who can then review objects of interest (OOIs) located in the biological specimen 12. The OOIs are arranged in a number of fields of interest (FOIs) that cover portions of the slide 14, so that the cytotechnologist's attention can be subsequently focused on OOIs within the FOIs, rather than slide regions that are not pertinent. The system 10 can be used for the presentation of cytological cervical or vaginal cellular material, such as that typically found on a Pap smear slide. In this case, the OOIs take the form of individual cells and cell clusters that are reviewed to check for the possible presence of an abnormal condition, such as malignancy or pre-malignancy.
The biological specimen 12 will typically be placed on the slide 14 as a thin cytological layer. A cover slip (not shown in FIG. 1) is preferably adhered to the specimen 12 to fix the specimen 12 in position on the slide 14. The specimen 12 may be stained with any suitable stain, such as a Papanicolaou stain.
An imaging station 18 is configured to image the slide 14, which is typically contained within a cassette (not shown in FIG. 1) along with other slides. During the imaging process, slides are removed from the respective cassettes, imaged, and returned to the cassettes in a serial fashion.
One known imaging station 18 includes a camera 24, a microscope 26, and a motorized stage 28. The camera 24 captures magnified images of the slide 14 through the microscope 26. The camera 24 may be any one of a variety of conventional cameras, such as cameras that can produce a digital output of sufficient resolution to allow processing of the captured images. A suitable resolution may be 640×480 pixels. Each pixel can be converted into an eight-bit value (0 to 255) depending on its optical transmittance. A value of “00000000” or “0” is the assigned value for least amount of light passing through the pixel, and a value of “11111111” or “255” is the assigned value for a greatest amount of light passing through the pixel. Thus, a “0” value indicates a dark value, e.g., a pixel of a fiducial mark, and a “255” value indicates a light value, e.g., an empty pixel.
The slide 14 is mounted on the motorized stage 28, which scans the slide 14 relative to the viewing region of the microscope 26, while the camera 24 captures images over various regions of the biological specimen 12. The motorized stage 28 tracks the x−y coordinates of the images as they are captured by the camera 24. Encoders (not shown) can be coupled to the respective motors of the motorized stage 28 in order to track the net distance traveled in the x- and y-directions during imaging.
Referring to FIG. 2, x−y coordinates tracked by the stage 28 are measured relative to fiducial marks 16 affixed to the slide 14. A fiducial mark 16 may be a rectangular patch of paint; in this case, one corner of the mark may be considered to be the marks's location. These fiducial marks 16 are also used by the reviewing station 22 to ensure that the x−y coordinates of the slide 14 during the review process can be correlated to the x−y coordinates of the slide 14 obtained during the imaging process.
More particularly, each reviewing station 20 includes a microscope 38 and a motorized stage 40. The slide 14 (after image processing) is mounted on the motorized stage 40, which moves the slide 14 relative to the viewing region of the microscope 38 based on the routing plan and a transformation of the x−y coordinates of the FOIs obtained from memory 36. These x−y coordinates, which were acquired relative to the x−y coordinate system of the imaging station 18, are transformed into the x−y coordinate system of the reviewing station 20 using the fiducial marks 16 affixed to the slide 14 (shown in FIG. 1). In this manner, the x−y coordinates of the slide 14 during the reviewing process are correlated to the x−y coordinates of the slide 14 during the imaging process. The motorized stage 40 then moves according to the transformed x−y coordinates of the FOIs, as dictated by the routing plan.
While known fiducial marks and coordinate systems used during imaging and review processes have been used effectively in the past, they can be improved. In particular, it can be difficult to locate fiducial marks in the presence of air bubbles and to focus on fiducial marks in the presence of dust and debris, as shown with reference to FIGS. 3-8.
FIG. 3 is a top view of a specimen slide 14 having three fiducial marks 16. A cover slip 50 is placed over the specimen 12. FIG. 4 is a top view of the slide 14 shown in FIG. 3 having dust or debris (generally dust 52) on the cover slip 50. FIG. 4 also illustrates an air bubble 54 underneath the cover slip 50. FIG. 5 is a side view of FIG. 4, which further illustrates dust 52 on top of the cover slip 50 and an air bubble 54 between the top of the slide 14 and the bottom of the cover slip 50.
Persons skilled in the art will appreciate that the dimensions shown in FIGS. 2-5 and other figures may not reflect actual dimensions and may not be to relative scale and are provided for purposes of illustration.
Referring to FIG. 6, when a cover slip 50 is placed on a slide, one or more air bubbles 54 may be trapped in the mounting medium 60 between the cover slip 50 and the slide 14. The fiducial mark 16 may appear to be a different shape and its true outline may be difficult to locate when an air bubble 54 or cellular debris overlaps the fiducial mark 16.
In addition, assuming a fiducial mark 16 is located, dust and debris 52 on top of the cover slip 50 may cause focusing errors. Automatic focusing on a specimen is generally done by focusing up and down until the objects in the image are in focus. Referring to FIG. 7, dust 52 or other debris (such as a fingerprint) on top of the cover slip 50 may cause an automatic focusing system or algorithm to focus on the dust 52, thereby resulting in a false plane 72, rather than locating the correct focal plane 70 corresponding to the sample 12 on the slide (which is coplanar with the fiducial marks 16).
The dotted line 80 in FIG. 8 shows focus or sharpness values for a set of images taken of a single microscope field at different focal heights. The field contains a fiducial mark 16 and also contains dust 52. The “x” axis represents a vertical distance (in microns) from the first or true focus plane 70. The second, false focus plane 72 is about 110 microns higher than the first focus plane 70; the false plane corresponds to the dust 52, which rests on top of the cover slip 50, which is about 100 microns thick. The “y” axis represents the logarithm of the Brenner score of the image. The Brenner score is a known method of quantifying image sharpness, and is the sum of the squares of the differences between the gray value of each pixel and its neighbor two pixels to the left, where differences less than a certain threshold are excluded from the sum to reduce the effect of image noise. A higher “y” axis value indicates that the image is in better focus and is sharper or clearer compared to lower “y” axis values. In the illustrated example, an automatic focusing system or algorithm that seeks to maximize the Brenner score would select the false focal plane 72, because its score is higher than the true focal plane 70.
Consequently, an imaging microscope 26 may focus on the dust 52 in the false focal plane 72 rather than on the fiducial mark 16 at the true focal plane 70. If the imaging station 18 scans this false focal plane 72 for cells instead of the true focal plane 70, many images taken of the sample 12 will be out of focus and objects of interest will be missed by the imaging software.
Thus, it would be desirable to have methods and systems that can more effectively locate fiducial marks on a specimen slide in the presence of air bubbles or other debris under the cover slip and that can focus on located fiducial marks in the presence of dust and debris on top of the cover slip.